Figure 1.
DLP lobeexhibits the primary changes in the PAP−/− mouse prostate.
The panels show an overview of the 12-old mice prostate dissected lobes. The DLP, AP and VP lobes were dissected from WT and PAP−/− mouse. The monolayer epithelium (white arrows) is seen in all the lobes of the WT mouse, whereas in the PAP−/− mouse an increased amount of cells is present in the lumen of the DLP lobe (black arrows). The AP and VP of PAP−/− mouse show no significant changes. Scale bars: 100 µm.
Figure 2.
PAP−/− mice develop prostate adenocarcinoma.
A, age related histological changes in PAP−/− mouse DLP. Epithelial hyperplasia was present in DLP of 3 month-old PAP−/− mice. The lumen was filled with dysplastic epithelial cells, and mPIN structures were observed in 6 month-old animals. Bulging of epithelial cells into the stroma (black arrows) through loosen fibromuscular sheath and prostate adenocarcinoma were present in 12 month-old mice. Scale bars: 100 µm. (n = 8). B, morphological abnormalities present in 12 month-old PAP−/− mouse prostates. Glands were filled with epithelial cells (black arrow head). Dyscohesive cells with double nuclei were present (white arrows), as well as sites of microinvasions of hyperchromatic epithelial cells with prominent nucleoli (black arrow). Cribriform structures (white arrowhead) and blood vessels among neoplastic epithelial cells (*) were also observed. Scale bars: 100 µm. (n = 8). C, 24 month-old PAP−/− mouse DLPs. Cells invaded the surrounding stroma and inflammation is present. Microinvasion of cells into the stroma and bulging is clearly observed (black arrows). Scale bars: 100 µm. (n = 8). FS: fibromuscular sheath, L: lumen, mL: monolayer epithelium.
Figure 3.
The prostate adenocarcinoma in PAP−/− mice is also detected by immunohistochemistry and electronmicroscopy.
A, smooth muscle actin (SMA) immunohistochemistry in 12 month-old mice. Monolayer epithelium (mL) and open lumen in PAP+/+ DLP. White arrows show the broken fibromuscular sheath (SM, smooth muscle) and bulging of epithelial cells to the stroma. Prostate adenocarcinoma (black arrows) is present in AP and DLP, showing a multilayer epithelium (ML) and inflammatory cells (black arrowhead) spreading in neighboring areas. Scale bars: 100 µm. (n = 4, per group). B, ultrastructural changes in 3 month-old and 12 month-old PAP−/− mouse DLPs. Monolayer epithelium, regular basement membrane (BM) and apical secretion are clearly seen in PAP+/+ mouse DLPs. 3 month-old PAP−/− DLPs show irregular BM and numerous apical vacuoles (red arrow head), as well the presence of basal lysosomes (Ly). In 12 month-old PAP−/− mouse DLPs, the epithelium has transformed to a multilayer epithelium containing hyperchromatic nuclei with multiple nucleoli. Pseudolumens (pL) have formed as a result of the growing and fusion of the epithelium. Invaginations of BM (red arrows) into the epithelium and numerous vesicles in the basal side of the cells (blue arrow heads) were additional signs of the transformation in the cells. Scale bars: 2000 nm (n = 4, per group).
Figure 4.
PAP−/− mouse DLPs release exosomal-like microvesicles.
A, electronmicroscopy images show the presence of electron-dense (white arrow) and electron-lucent (black arrow) microvesicles (∼30 to 80 nm) in the lumen of the acini, and MVE containing microvesicles in the apical part of the cell. Scale bar: 1,000 nm. B, numerous microvesicles are present in the apical region of PAP−/− DLP and secreted into the lumen, decreased amount of microvilli is observed (black arrowheads) (scale bar: 1,000 nm). C, microvesicles are secreted into basolateral intercellular space of PAP−/− DLP (scale bar: 2000 nm). D, lamellar body-like structures (*) are inside the epithelial cell (scale bar: 500 nm) and E, released into the lumen (*). Scale bar: 200 nm. F, TMPAP and snapin are also present in exosomes. Immunoblots of exosomes isolated from TMPAP/LNCaP cell culture medium. Flotillin and CD13 were used as exosomal and prostasomal marker respectively.
Figure 5.
Proliferation of DLP cells is increased in PAP−/− mice compared to WT mice.
A, bar plot showing the ratio (as percentage) between proliferative cell counts and total amount of cells. (**, P value <0.01; ***, P value <0.001). Error bars indicate S.E.M. values. B, bar plot showing the ratio (as percentage) between apoptotic cell counts and total amount of cells. Error bars indicate S.E.M. values.
Table 1.
Significant ontological terms obtained with GoMiner software from two-color microarrays experiments.
Figure 6.
TMPAP co-localize and interact with snapin in the cell lamellipodia.
A, co-localization (yellow) of TMPAP (green) with snapin (red) was observed in the vesicles and lamellipodia of the TMPAP/LNCaP cells. Arrows mark the co-localization points in the upper panel (scale bar: 20 µm). Lower panel (scale bar: 3 µm) showing the lamellipodia region, amplification of the area marked with a box in the upper panel (left). B, intensification of donor (TMPAP-GFP) fluorescence in LNCaP cells was observed after acceptor (snapin-DsRed) photobleaching which confirms FRET between two molecules (Scale bar: 2 µm).
Figure 7.
TMPAP is involved in endo-/exocytosis (proposed mechanism).
TMPAP synthesized in the endoplasmic reticulum is transported in vesicles to the plasma membrane through the trans-Golgi network (TGN). After the vesicle docking and fusion events leading to release of vesicle content, TMPAP inserted in plasma membrane exerts its phosphatase function over AMP. The resulting product adenosine (Ado) activates the adenosine receptors, which are GPCRs, A1 or A3with Gαi (inhibitory G-protein β-subunit) specificity leading to the inhibition of adenylate cyclase (AC) activity, and A2 adenosine receptors with Gαs (stimulatory G-protein α-subunit) producing the stimulation of AC activity. Activated AC produces cAMP, which activates PKA responsible for the phosphorylation of snapin. The turnover is completed by clathrin-mediated endocytosis of SNARE components and TMPAP for recycling and degradation in lysosomes vía the endosomal-lysosomal pathway. From early endosomes, the cargo can be sorted to late endosomes or to MVE, which can follow the route leading to exosome release. Additional dephosphorylation events by TMPAP can occur while trafficking between different compartments. From late endosomes, TMPAP can go to lysosomes or back to TGN via the retrograde pathway. ATP: adenosine triphosphate, ADP: adenosine diphosphate, AMP: adenosine monophosphate, Ado: adenosine, TGN: trans-Golgi network, P: phosphate group, AP-2: adaptor protein complex 2, ADORA: adenosine receptor A (types A1, A2 and A3), AC: adenylate cyclase, Gαs, Gαi, Gβ, Gγ: G-protein subunits, VDCC: Voltage-gated calcium channel. Synaptobrevin, syntaxin and SNAP25 are SNARE proteins. PI (4,5) P2: phosphatidylinositol 4,5-bisphosphate.